One of most important factors in establishing a viral infection is the presence or absence of particular receptor molecules. These proteins/sugar molecules (or whatever) must be expressed on the surface of the cell, allowing the virus to bind and initiate entry.

The expression of a receptor therefore governs the behavior of an infection by allowing only certain cells to permit virus replication. This thus controls how a virus enters your body, spreads throughout it, causes disease and finally escapes to continue the infection in a new host. The identity of the receptor that a virus uses allows a better understanding of infection and pathogenesis and may facilitate the development of new antiviral treatments.

A paper published in PLoS Pathogens (see here), this week documents the discovery that a protein - Nectin-4 - acts as the receptor that may allow measles virus (MeV) to escape your body in the ends-stages of the disease, through the infection of the epithelial cells lining your respiratory tract. However, this molecule differs from that used bythe virus to initially enter your body.

This Nectin-4 study finally sheds light on what has been an elusive molecule to find and goes a long way in explaining the basic biology of this historic human pathogen. Also, the findings that Nectin-4 is highly expressed on the surface of a number of common cancers further adds weight to the use of MeV as an anti-cancer agent. But does Nectin-4 fully explain MeV infection? And, can we finally close the book on measles infection?

Despite the widespread use of a highly effective - and safe - vaccine, MeV still infects an estimated 20 million people a year, mostly young children and, in 2008, the WHO documented that measles caused the death of 164,000 of these children that year. Measles is characterised by fever, photophobia, cough, runny nose, sickness, and a nasty rash over most of the body (see picture above). Every so often the virus will enter your brain and can cause serious inflammation there. And, even more rarely will the virus persist in your body only to re-emerge years later as an incurable degenerative brain disease known as sub-acute sclerosing pan-encephalitis (SSPE). The molecular basis of how this virus enters our body, replicates, spreads inside you exits your body are only now being realised with modern recombinant viruses, like those expressing fluorescent proteins (see below). For examples, see here, here, here and here.

Disclaimer: these references are from work that my supervisor and the group I am currently in are heavily involved in but are the only work documenting MeV in the entire organism. Read here when I previously wrote about the discovery of how MeV enters our body through dendritic cells.

It was always assumed that following breathing in virus-laden aerosols MeV initially infected your epithelial cells lining the upper respiratory tract. From here it could spread throughout your body, replicating in lymphocytes and finally be released back out via the epithelial cells. But, researchers using a green-fluorescent protein (GFP)-expressing MeV showed that it was in fact dendritic cells (sentinal immune cells) found within your respiratory tract that were the first cells to be infected. These cells could then easily transmit the virus to your immune cells. These cell types can be justified through the identify of the receptor for measles, SLAM that is only expressed on these immune cells.

However, based on animal models and autopsy reports, we know that the virus can - and does - productively infect epithelial cells lining your airway (see figure above) yet no receptor was known. That is, until Chris Richardson's team at Dalhousie University, Canada discovered that Nectin-4 could function as a MeV epithelial receptor.

PVRL4 (Nectin-4) allows MeV infection

The group used microarray analysis to determine what genes were expressed in cells that could, and those that could not, be infected by a wild-type, pathogenic MeV.

They were then able to bioinformatically pull out proteins that were found on the plasma membrane surface (ones that were likely to be receptors) and expressed them in cells that couldn't be infected. The gene that allowed MeV to infect was the receptor molecule and this was confirmed to be Nectin-4 through siRNA knockdown and antibody binding assays.

So does Nectin-4 explain how MeV infects, causes disease and escapes the human body? Based on the Human Protein Atlas entry for Nectin-4, this protein is on most normal cells weakly - including the brain and lung, which are both targets for MeV and it is not really expressed on lymphoid cells, although these cells already express a highly-active receptor for the virus. This study may explain how the virus can infect your respiratory tract epithelium and be secreted into your airways to spread the infection. The role of Nectin-4 should be established in further model systems like the macaque and even in vitro differentiated epithelial cells.

The sheer amount of genomic data now available from a wide range of species has allowed the increased scrutiny over what genes and DNA sequences are present in their chromosomes. What we have begun to notice is that many of these sequences have a viral origin.

And, in the recent half-decade, the numbers of these endogenous viruses discovered have rapidly increased, but how did they get there? What are they doing? And, are they bad for us? Only a true experimental model system can answer these question but this is something which is lacking.

Lets talk about ERVs

Now, viruses have left their mark on our genomes in more ways than one; infection and associated disease/mortality has heavily influenced the genetic structure of populations via natural selection and genetic drift for millions of years. Yet, another important mechanism is that employed by the endogenous retroviruses (ERVs) that have inserted a DNA copy of themselves into our chromosomes - the norm for retroviruses - and have forever become part of us.

Over the course of evolution, these once infectious viruses have become redundant, building up a collection of genetic mutations resulting in loss of replicative ability. Although many still play a role in the cellular biology of the host and have been a great source of genetic novelty over the billions of years of evolution.

However, what we have noticed is that viruses other than retroviral species have inserted themselves into genomes of humans, other animals and even plants and fungi. Many of these viruses have a DNA phase in their replication cycle, which is put into the genome of their host to aid their survival and so it may not be all that surprising that they have stayed with us through evolution (these viruses include many single-stranded DNA viruses and again).

One intriguing observation is that many of these non-retroviral endogenous viruses are in fact - or were - RNA viruses with no known DNA phase during replication. They are therefore called Non-retroviral RNA virus sequences (NRVSs). See plant NRVSs and mammalian NRVS (ebola virus-like , borna virus-like and many more - (lots, they're everywhere). There is strong evidence that these integrations occurred thousands, if not millions of years ago and could have played a role in the evolution of many species.

How can we study these viruses?

But just exactly how do these viruses do it? After-all, they are RNA viruses without a reverse-transcriptase enzyme and hence no natural ability to produce a DNA genome that can be inserted into our chromosomes. And, can we follow this endogenisation experimentally? One mechanism is thought to occur when an endogenous retrovirus-like element joins itself to a non-endogenous RNA virus and then this chimera is put into our genome. But this is really only half the story - can we ever study the entire process, from initial infection to endogenisation?

For an RNA virus to become fully integrated into our germline it has to first infect our germ-line cells (sperm/oocytes); its RNA genome must be copied into DNA and this DNA molecule must be inserted into the chromosome. It also must allow for the development of healthy and reproductively active offspring and can then let evolution take its course. An experimental model system of this process would allow for a better understanding of this process in molecular detail and how this relates to the evolutionary process as a whole.

Here's how you would do it:

The animal model

Bank vole - a good model for endogenous viruses?

A small-animal model that could be infected by a type of virus that had been shown to integrate into the genome (borna disease virus, for example) would make this easier to study. Plus, many rodents have been shown to harbour many NRVSs already.

The virus infection

You would infect the animals with the virus in as natural conditions as possible and look to see whether the virus entered and replicated in the cells of the germ-line.A GFP-expressing virus would work best for this.

Detection of RNA - DNA

What you would have to do is be able to track the process of turning the RNA genome into DNA. A PCR-based screening would work well for this and could be applied to a range of tissues in the host, including occytes/spermatozoa.

Integration

To prove that the DNA copy was inserted into the host chromosome you would need to sequence the sites where the DNA had integrated in and determine where in the genome it lay.

Stability

This experimentally infected rodents could be bred continously and the presence of endogenised virus looked for in their offspring. The expression of said virus genes (if there is any) could be followed in rodent tissues.

Well one paper has maybe taken the first step in the development of such a model system (although they may not know it). It has shown evidence that if you infect baby bank voles with borna virus, directly into their brain you can detect borna virus-specific DNA sequences using PCR following DNA extraction (see above PCR gel for results). And, these sequences resulted from the virus, not some already-endogenised borna virus sequence. Although they did not check for germ-line infection or integration, this is the first step. The applicability of Borna virus reverse genetics and these animal models could make this kind of study feasible but certainly not easy. We may in future catch a glimpse of this process in real-time.

Our struggle with HIV/AIDS epitomizes societies' millennia-old fight with microbial pathogens. One goal of HIV research is to generate effective interventions that will allow us to: 1) prevent further spread of HIV (vaccines and behavioural changes), and 2) eliminate the virus from those already infected (antivirals).

This sterilizing immunity - as it is referred to - has been a long sought-after goal for a number of viruses, yet widespread use of highly-active anti-retroviral therapy (HAART) fails to completely remove the virus from an individual patient. Somewhere, somehow HIV is continuously replicating in your body. But how and why is this possible?

The spread of HIV from Dendritic cells to T lymphocytes or T to T cells may allow escape from antivirals. http://pathmicro.med.sc.edu

One potential mechanism is that HIV lies in a latent state - anatomical (central nervous system) or biochemical (following DNA integration and before gene expression) - where these drugs cannot inactivate it.

But researchers, headed by David Baltimore at the California Institute of Technology, have come forward with both theoretical and experimental evidence suggesting a novel mechanism that explains how the virus may be able to circumvent HAART treatment through continuous replication and direct cell-cell spread. Their results were published in Nature last week. See here.

What is cell-cell spread?

Viruses can infect new cells via a number of mechanisms. The most well-characteristic being via cell-free virus particles (see computer simulation paper here). In this, new viruses are released from the originally infected cell and diffuse to an uninfected one nearby, thus establishing a novel infection.The problem with this is that it is pretty inefficient. For one, the viruses could be carried anywhere and even if they reach a cell, it may not be the right one. One other, more efficient means of transmission is through direct cell-cell spread, which generally takes diffusion out of the picture.

Following interaction with dendritic cells or T cells, the virus directs the assembly of a structure (referred to as the virological synapse) linking the two cells via alterations of the cytoskeleton which causes the two plasma membranes to come close together. This structure initially derives from the target cell.

It is here where new virus particles are released into the gap that has formed, thereby increasing the efficiency of spread by directing transmission and limiting the effects of diffusion.This synaptic structure, ultimately acts to concentrate both virus and receptors at defined subcellular locations. This kind of spread may allow the virus to become essentially invisible to our bodies' immune defences, such as neutralizing antibodies, and even antiviral drugs.

How does this allow escape from treatment?

Their model: a) inefficient cell-free infection will be eradicated with antvirals. The more efficient direct cell-cell spread will persist as it involves many more virus particles. b) The mathematical model (backed up by experimental data) showing the loss of infection (transmission index) with increasing concentration of antiviral (TFV) when we have few (m =0.2) or many (m = 100) virus particles.

Expanding the model

Initially, they generated - and experimentally verified - a mathematical model of how HIV transmission may differ during antiretroviral treatment under cell-free (inefficient - low virus transmission) or direct cell-cell spread (efficient - high virus transmission) mechanisms. Their results indicate that when there are many viruses around, the infection is more resistant to the drugs. This, they suggest, is due to the increased probability that at least one virus particle will not interact with the antiviral.

This observation extended to direct cell-cell spread when they introduced previously infected cells with non-infected ones and measured transmission of virus. Both the experimental and theoretical modelling suggest that this spread could be responsible for the ongoing replication seen in patients being treated with antivirals.

What does this mean for HIV therapy? Well, this work brings experimental evidence on the already known process of ongoing HIV replication in the face of antivirals. Through experimentally identifying this mechanism, the researchers here have uncovered a weak-point in HIV biology, one that is not currently being targeted. This method of cell-cell spread may also facilitate escape from neutralizing antibody responses through physically preventing their interaction with HIV proteins. How then might our own bodies combat this virus if its ability to more from cell-cell was inhibited? This work further adds to the growing body of work highlighting direct cell-cell spread as a principle mode of transmission for these retroviruses.

Everyone knows the 'flu when they see it. Its like the cold but only worse, right? But often the public are very quick to brand something the flu when it clearly isn't and this even extends to doctors in the clinic. As highlighted by this recent paper, published in BMC Infectious Diseases.

Turns out a lot of viral infections look like influenza, clinically. But what are doctors misdiagnosing as flu? Based on this study, which paired molecular diagnostics with what the clinic thought each patient had based on well-defined guidelines (mostly flu at the early stages of the 2009 swine-influenza pandemic - April - July).

Out of the 3247 samples analysed, 27.9% were diagnosed as being infected with a respiratory pathogen they looked for, what is causing their symptoms??. While human rhinovirus infections were the most common, H1N1 influenza infection was one of the least common at 0.8%. A whole heft of viral and bacterial pathogens were detected at levels intermediate to these.

So, whats going on here? How come it's so difficult to diagnose these guys and after-all, does it really matter?

Well, this work highlights the difficulty in accurately determining the causative pathogen from basic symptoms. The viruses looked at in this study all share replicative and epidemiological characteristics: respiratory pathogens, infect the upper/lower respiratory tract and, as viruses, induce a general interferon/inflammatory response (sore throat, headache, malaise, fever, muscle pains etc.) and are transmitted among the winter/spring months. It is not until you look at the molecular level, that you realise they are all at least slightly different.

And, yes it does really matter. Many of these people may have developed life-threatening symptoms as a consequence of an infection that was not influenza and would have been missed by the clinicians. Also, at a great cost to society and the patient (financial, medical and emotiona;), some people were wrongly treated with antiviral medication after being diagnosed swine flu.

What studies like this show, is the importance of molecular diagnostics in times of pandemic infections. But the problem here is that, despite their high specificity, they can be expensive and difficult to run, especially in 'field' locations across the world.

You may have read earlier this week how human cytomegalovirus - a seriously common pathogen - was discovered to be a possible cause of high blood pressure. What you may not have heard about (or read), is the actual paper that these headlines refer to - see paper here. And, if you had, you may not have been so quick to jump to that same conclusion the media had for it was only a correlation - no causation was determined - but the results are nonetheless important, as you'll see below.

Hypertension - or high blood pressure - is a chronic disease with deadly implications, affecting an estimated one billion adults worldwide. Referred to as a global pandemic, if left unchecked, high blood pressure can result in terminal damage to both blood vessels and organs leading to stroke, heart attack or kidney failure. And, as if this weren't bad enough, it kills silently while if detected early enough it can be effectively controlled through various drug regimes and lifestyle changes.

Two flavors of hypertension are currently recognized: essential - the most common sort (90%) and secondary, the more rarer form. The causes of secondary are well characterized and usually result from a number of acute illnesses, while the origins of essential are more difficult to determine. This most likely reflects the complex nature of the disease, which results from interactions from both genetic and environmental components; any one of these in isolation having little effect on its overall state.

Given its global importance, the factors predisposing to - or causing - essential hypertension are subject to intense research, making the identification of a viral origin of the disease all the more interesting.

HCMV, a member of the herpesvirus family of viruses (large, double-stranded DNA viruses with over one hundred genes) is an expert human pathogen. And, with 40% percent of the human population estimated to be infected with the virus, who can argue. Also based on seroprevalence, 80% of the elderly population are infected making it one serious human pathogen.

Following initial infection, HCMV has the ability to rapidly enter a latent state, persisting within specific cells inside your body. The usual cells are fibroblasts, endothelial cells and some white blood cells. From here, it will every-so-often re-activate itself and be shed in your saliva, ready to infect the next susceptible person. So once you get infected, you are always infected. Although it barely causes disease in those with fully functioning immune systems, the people who are immuno-compromised (neonates, AIDS sufferers, chemotherpy patients or the elderly) aren't so lucky, giving rise to calls to develop a vaccine.

How does one relate to the other?

Originally carrying out an investigation into the gene regulatory microRNA (miRNA) expression differences between Chinese patients with and without high blood pressure, the researchers soon discovered an obvious sign of HCMV infection in the form of a virally encoded miRNA.

Plasma miRNA expression levels in the Chinese study. Note the number of differentially regulated miRNAs. The group focus on hcmv-miR-UL112 (first from top left) although others could have been addressed.

Quantitatively determining the miRNA levels in blood samples, many miRNAs were up or down regulated but the group focused in on one in particular: hcmv-miR-UL112 - derived from HCMV that was shown to be highly expressed in those suffering from primary hypertension. These expression differences were thought to be down to endothelial cells within the blood sample, termed circulating endothelial cells. HCMV seropositivity as well as higher amounts of viral DNA, in general, was seen to be correlated with the high blood pressure group, especially when other factors were considered. This correlation was not seen with other common human viruses (Epstein-Barr and adenoviruses) suggesting a certain specificity.

How might HCMV cause hypertension?

Well this paper doesn't prove that one causes the other, only that HCMV infection and hcmv-miR-UL112 expression in particular significantly correlates with high blood pressure. There of course may and will be other factors influencing this, but in speculation, how may this miRNA influence disease pathology?

As I mentioned before, HCMV lies dormant inside the endothelial cells lining your blood vessels. Although latent, a number of genes are expressed from its genome specifically hcmv-miR-UL112, which helps it avoid detection by our bodies immune system. The researchers in this paper demonstrate an additional target for this molecule in Interferon Regulatory Factor 1 (IRF-1) a key regulator of the immune response and has been implicated in a number of vascular diseases (they list a dozen hypothetical mechanisms linking HCMV to hypertension.)

Although this paper did not provide conclusive evidence that HCMV causes high blood pressure (Koch's postulates were not fulfilled or even looked at), it does shine some light on the causes of essential hypertension - a long sought after goal. Further studies will need to validate these results in non-Chinese samples and investigate mechanisms in which latent and/or replicating HCMV can influence endothelial functioning in vivo.

Any intelligent fool can make things bigger, more complex, and more violent. It takes a touch of genius -- and a lot of courage -- to move in the opposite direction.

E. F. Schumacher (1911-1977) British Economist

Vaccines represent one of the most cost effective methods around to prevent loss of life and disease in a whole range of animals, including the human population. Over the last 200 years or so, we've become pretty adept at producing them and so the science of vaccinology - or how to generate these complex pharmaceuticals - has led to the eradication (and near eradication) of many viral pathogens.

This is one network in your immune response following influenza vaccination (Kokke et al 2011) as id'd through systems biology approaches. Knowledge of key mediators in these pathways may allow for the rational design of new vaccines - but is it worth it?

But, it hasn't succeeded for a number of currently killer viruses (respiratory synycitial virus and HIV to name but two) and we have begun to think that maybe the method of 'isolate, attenuate, vaccinate' or the synthesis of single virus antigen molecules isn't gonna cut it anymore. So what are we going to do?

We are wanting to rationally generate vaccines - taking a wild-type, disease-causing isolate and through some genetic engineering, make it sufficiently weak so as to generate an effective immune response in patients while not causing disease. Yet, this is harder than it looks and so some researchers are now turning to systems biology to offer a glimpse into how some of our most successful vaccines function so as we can reconstruct these processes for the numbers of viral pathogens we are yet to protect against.

*I have explored how we may attempt to do this from the viruses perspective (mumps virus versus vaccine, here), more precisely: how come the vaccine strain is less deadly than the 'wild' strains. Both are valid approaches and probably just as difficult to carry out as each other.*

Systems biology affords us a chance to more fully understand the complexity of living systems. Through the collection of reams of data (DNA sequences, gene expression changes, protein levels - and other 'omics' technologies) we are now able to adequately model what is going on in the organism/cell through now more common bioinformatic and statistical analyses. As in the quote I used above, it is not about making the study of life more complex, it is really about realizing this fact and doing something to understand it better under a broader way of thinking. This allows us to 'see' changes and functional differences that we would never have observed had we gone about such an experiment using our a priori knowledge and this global, holistic view may just be the savior that vaccinology needs now.

Example of the complex data collected during a typical 'systems' experiment - what does it all mean, and how can we find something important and worthwhile to study?

I am aware of a number of papers that are currently using this process as a primer to develop improved vaccine products (see here, here and review here of virus vaccine examples). These guys - for example: Bali Pulandran of the Emory Vaccine Centre in Atlanta, USA - are interested in comparing the immune response (humoral response, innate immunity and gene expression changes) of human subjects administered with vaccines. The response to the yellow fever virus vaccine as well as two types of influenza vaccines have been approached and through complex bioinformatic modelling they were able to pull out some significant correlates of immune response - this they hope will aid in the future testing of novel vaccines and facilitate a rational take on vaccine generation by identifying a gene(s)/protein with a functional role in the immune response. This they have begun to do in some of ther papers above - it is nice to see this kind of work being used as a basis for experimental biology.

These types of studies hail a new way of thinking about viruses, vaccines and the immune response to them; if only we can realize the power in taking a step back and looking at the diversity in each. And this type of work could be applied to any number of mechanisms such as vaccine safety or applying it to different tissues during an infection.

Saying that - this stuff isn't particularly easy, cheap or quick as you might think. But as each study generates so much data, might it not take but a few such investigations to lead us on the way of rationally attenuated and protective vaccines? So, should vaccinology embrace systems biology? I think if you have the abilities to do such a study - which from a pharma perspective definitely yes, do it as the more information we have at our disposal the better position we are in. We await further results from these groups to compare how well systems thinking goes up against human ingenuity, that has worked well in the past.

Bring to mind the now famous 'first scientific exploration of vaccination', when, in the late 1700's, Edward Jenner - an English physician - first came up with the idea of using a non-pathogenic cowpox virus to vaccinate people against its deadly relative, smallpox (variola virus).

Well, this virus and others like it, such as vaccinia virus (and its own viral derivatives, like the highly attenuated modified vaccinia virus Ankara) have been used worldwide to protect human populations from contracting smallpox (See dryvax and it's recombinant clone ACAM2000), which resulted in the eradication of variola virus during the second half of last century. These viruses are thought to all trace there ancestry back to cowpox isolates from around Jenner's time in the late 1700's, yet we don't really know for 100% where they originated.

Despite its renowned success, showing society the power of vaccination, the origins of cowpox have so far remained elusive. The story goes that Jenner's original cowpox isolate, through generations and generations has somehow become what we know of as vaccinia virus but how is anyone's guess. Maybe recombination with other poxviruses out there or maybe it is the last living representative of an extinct virus is the reason why.

What human cowpox looks like

Now, an international team of researchers (see paper here) has shed light on it's origins by sequencing and studying whole genomes - contrary to the single-gene-centric studies in the past - of multiple currently circulating isolates of cowpox from around the world in order to uncover the secrets of poxvirus evolution in general.

Viruses are a haven of genetic diversity - even DNA viruses, which have been largely ignored on that front in favor of their more mutation-prone RNA cousins; this fact is no more apparent than in the case of poxviruses. These viruses, including smallpox and the re-emerging human pathogen monkeypox represent an immense amount of genotypic and phenotypic variation, which is in itself medically and evolutionarily important. Just think of the devastation that smallpox caused to the human population and have a look at what monkeypox has been up to.

False-color electron micrograph of vaccinia virus particle

This group compared the DNA of the cowpox strains to other closely related pox viruses, such as: smallpox itself, monkeypox, camelpox and tatera pox (viruses which themselves have a difficult to trace past) as well as current vaccine vaccinia strains. They thus generated large phylogenetic trees based on the compared sequence and then mapped these onto a map of Europe to see if they could uncover some geographical pattern of cowpox evolution.

This analysis found an as yet unappreciated diversity hidden under what we called 'cowpox viruses' through identification of a number of well-defined monophyletic groups that should in their own rights be designated separate species. Interestingly, it also appears that our vaccine strain has jumped species to horses and buffalo as viruses isolated from these species have close relatives in vaccinia-like strains.

Cowpox viruses were found to cluster in two major groups - cowpox like and vaccinia virus like suggesting that our smallpox 'vaccinia' vaccine potentially originated as a cowpox virus (as we thought) yet it was endemic to mainland Europe, something that goes against the tale of Jenner's isolation of cowpox from the UK.

The authors suggest that further sampling of more isolates from within the UK and across Europe may clear up any taxonomic uncertainties here. What this work does highlight is the oft under appreciated diversity of large DNA viruses, especially the medically important pox viruses and the difficulties of doing evolutionary analysis on viruses which such large genomes who like to recombine with each other.

About Connor

Connor Bamford is a PhD student at a university in Northern Ireland, UK. He is studying the molecular biology behind how viruses infect and cause disease in humans. DISCLAIMER: the views expressed in this blog are my own and are not to be confused with those of my institution where I study, my supervisor's, my group's, my nationality, my ethnicity, my gender nor my starsign.